Commercially viable process for in vitro mass culture of jatropha curcas

ABSTRACT

The present invention relates to a commercially viable process for in vitro mass culture of  Jatropha curcas . The process for in vitro mass culture of  Jatropha curcas  is simple, faster, and suitable for production of disease-free root tubers of uniform quality and employs media with a reduced concentration of phytohormones.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 11/706,608, filed Feb. 24, 2007 the contents ofwhich is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods for in vitro micropropagationof Jatropha Curcas. The invention in particular relates to acommercially viable process for in vitro mass culture using media with areduced concentration of phytohormones.

BACKGROUND INFORMATION

Jatropha curcas, belonging to the family of Euphorbiaceous, is a plantof Latin American origin, widely spread throughout the arid andsemi-arid tropical regions of the world. Jatropha is a large genuscomprising over 170 species. The most common species in India are J.curcas, J. glandulifera, J. gossypifolia, J. multifida, J. nana, J.panduraefolia, J. villosa and J. podagrica.

J. curcas is a small tree or shrub with smooth gray bark, which exudeswhitish colored, watery, latex when cut. Normally, it grows betweenthree and five meters in height, but can attain a height of up to eightor ten meters under favorable conditions. It is a drought-resistantplant, living up to 50 years and growing on marginal lands.

J. curcas has large green to pale green leaves, which are alignedalternate to sub-opposite. The leaves are three-five lobed with a spiralphyllotaxis. The petiole of the flowers ranges between 6-23 mm inlength. The flowers are formed in hot seasons. Several crops are formedprovided the soil is moisture is good and temperatures are high. Inconditions where continuous growth occurs, an imbalance of pistillate orstaminate flower production results in a higher number of femaleflowers. Fruits are produced in winter when the shrub is leafless. Eachinflorescence yields a bunch of approximately 10 or more ovoid fruits.Three, bi-valved cocci are formed after the seeds mature and the fleshyexocarp dries. The seeds become mature when the capsule changes fromgreen to yellow, after two to four months from fertilization. Theblackish, thin-shelled seeds are oblong and resemble small castor seeds.

This plant has various medicinal uses especially in nutraceuticals,pharmaceutical, dermatological, and personal care products. The latex ofJatropha curcas has anticancer properties due to the presence of analkaloid known as “jatrophine.” The tender twigs are used for cleaningteeth. The juice of the leaf is used for external application for piles.The roots are used as an antidote for snake-bites. The seeds are usedfor antihelmithic purposes.

The bark yields a dark blue dye used for coloring cloth, fish net andlines.

Most of the Jatropha species are ornamental except for J. curcas and J.glandulifera which are oil-yielding species (as projected inpresentation on “Biotechnological interventions for production andplantation of improved quality of Jatropha” by Dr. Renu Swarup, 2004).The seeds of these species contain semi-dry oil which has been founduseful for medicinal and veterinary purposes (Gubitz et al., 1999“Esterase and lipase activity of Jatropha curcas seeds J. BiotechnologyOct 8:75(2-3): 117-26).

The oil content is 25-30% in the seeds and 50-60% in the kernel. The oilcontains 21% saturated fatty acids and 79% unsaturated fatty acids.Jatropha oil contains linolenic acid (C18:2) and oleic acid (C18:1)which together account for up to 80% of the oil composition. Palmiticacid (C16:0) and stearic acid (C18:0) are other fatty acids present inthis oil.

The oil is non-edible, however it has the potential to provide apromising and commercially viable alternative to diesel oil as it hasall the desirable physicochemical and performance characteristics asthat of diesel. The plant J. curcas has lately attracted particularattention as a tropical energy plant. The seed oil can be used as adiesel engine fuel for it has characteristics close to those of fossilfuel diesel. Moreover, due to its non-toxic and biodegradable nature,Jatropha biodiesel meets the European EN 14214 standards of a pure andblended automotive fuel for diesel engines. Jatropha curcas seed yieldsapproach 6-8 MT/ha with ca 37% oil. Such yield could produce theequivalent of 2100-2800 liters of fuel oil/ha, whose energy isequivalent to 19,800-26,400 kwh/ha (Gaydou, A. M., Menet, L.,Ravelojaona, G., and Geneste, P. 1982. Vegetable energy sources inMadagascar: ethyl alcohol and oil seeds (French). Oleagineux37(3):135-141).

Because of its very high saponification value and its ability to burnwithout emitting smoke, the oil of the seeds is commercially useful. Forexample, it is extensively used for making soaps.

Therefore, in view of the above, there is a need to provide method formicropropagation of Jatropha curcas which are economical and allowproduction on a commercial scale of uniform quality, true-to-type,disease-free plants.

Plant Tissue Culture

Micropropagation is the in vitro regeneration of plants from organs,tissues, cells or protoplast using techniques like tissue culture fordeveloping true-to type resultant plants of a selected genotype. Ingeneral, tissue from a plant commonly known as an explant is isolated tocreate a sterile tissue culture of that species in vitro. A culture isinitiated from an explant. Once a culture is stabilized and growing wellin vitro, multiplication of the tissue or regeneration of entire plantcan be carried out. Shoots (tips, nodes or internodes) and leaf piecesare commonly used but cultures can be generated from many differenttissues. Juvenile tissues generally respond best. Besides the source ofthe explant, the chemical composition of the culture medium and thephysical environment of cultures have been found to be of a greatinfluence on the regeneration capacity, multiplication ratio, growth anddevelopment of new plants in the culture system. Therefore one needs tooptimize these factors for each individual plant species.

Sujatha and Mukta (“Morphogenesis and Plant regeneration from tissuecultures of Jatropha curcas”, Plant Cell Tissue & Organ Culture,44(135-141) 1996) have reported a method for the differentiation ofadventitious shoots through callus derived from hypocotyl, petiole, andleaf explants of J. curcas. Weida Lu, Tang Lin, Yan Fang & Chen Fang(2003) (“Induction of callus from Jatropha curcas and rapidpropagation,”

College of Life Science, Sichuan University Chengdu 610064, China) havereported induction of adventitious buds and regenerated shoots fromepicotyl explants through callus.

All of the above studies focused on callus-mediated regeneration. Planttissue regeneration through a callus stage is vulnerable to somaclonalvariations and hence will not ensure true-to-type plants from elitemother plants. In addition, all of the above studies used non-meristemtissue, which is more likely to be infected with disease than meristemtissue. Therefore, there remains a need in the art for micropropagationmethods that allow the production of true-to-type, disease-free plants.

SUMMARY OF THE INVENTION

The present invention provides for the first time the use of meristemtissue as an explant for direct organogenesis giving rise totrue-to-type clones. Although Applicants do not wish to be bound bytheory, they believe that the success of the present invention dependsupon the use of meristem and a low concentration of phytohormones.

The invention provides methods for producing a true-to-type clone of aJatropha curcas mother plant by culturing a meristematic explant ofJatropha curcas in media with phytohormones at a concentration fromabout 0.01 mg/L to about 10 mg/L and producing a true-to-type clone of aJatropha curcas mother plant from said meristematic explant.

In preferred embodiments, the meristematic explant is from a shoot tipor a nodal bud. Preferably, the shoot tip has bud tissue. Preferably,this bud tissue is apical bud tissue.

In certain embodiments, the phytohormones are at concentration of 0.01mg/L, 0.1 mg/L, 0.5 mg/L, 1 mg/L, 5 mg/L, or 10 mg/L. Preferably, thephytohormones are at a concentration from about 0.1 mg/L to about 0.3mg/L.

The phytohormones used in the invention may be cytokinins,cytokinin-active urea derivatives, auxins, or gibberellins. Cytokininsthat may be used in this invention include 6-aminopurine (adenine),6-aminopurine hydrochloride, 6-aminopurine hemisulfate, 6-benzylaminopurine (BAP), kinetin, zeatin, and N6-substituted derivatives. Inpreferred embodiments, the cytokinin is 6-benzyl aminopurine at aconcentration from about 0.44 μM to about 2.22 μM and most preferably atabout 0.44 μM. Cytokinin-active urea derivatives that may be used inthis invention include thiadiziron, diphenylurea, andN-phenyl-N′-(4-pyridyl) urea. Auxins that may be used in this inventioninclude naphthalene acetic acid, naphthaleneacetamide, naphthoxyaceticacid, indole acetic acid, indole butyric acid (IBA),4-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), and2,4,5-trichlorophenoxyacetic acid. Preferably, the auxin is indolebutyric acid is at a concentration of about 4.9 μM.

Steps in in vitro mass culture can include selecting the healthy motherplants, isolating explants from a mother plant, cleaning, sterilizingthe explants by primary and secondary sterilization, inoculating theexplants on culture initiation medium having basal salts of MS medium togive multiple shoots, transferring the cultures to proliferation andelongation medium having basal salts of MS medium the same as theinitiation medium, transferring the elongated shoots to rooting mediumhaving basal salts of MS medium, subjecting in vitro grown plantlets toprimary and secondary hardening, and transferring the hardened plantletsto fields.

In preferred embodiments of this invention, explants are selected frombuds with shoot tips and nodal segments. In the most preferredembodiments the explant is the apical bud.

In the most preferred embodiments of the present invention the MS mediumemployed for culture initiation, proliferation and elongation, androoting has a reduced phytohormone level, thereby rendering the processcost-effective.

Thus, the use of meristematic tissue as an explants and the use of veryreduced concentrations of phytohormone, such as 0.44 μM-4.4 μM of6-benzyl amino purine in proliferation and initiation medium, which cangive 3-4 shoots per explants and high success rate during rooting andhardening, renders the present invention commercially viable for invitro mass culture of Jatropha curcas for large-scale multiplication oftrue-to-type clones of elite variety plants.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, the inventions of which can be better understood byreference to one or more of these drawings in combination with thedetailed description of specific embodiments presented herein.

FIG. 1 shows the initiation of the apical bud of Jatropha curcas.

FIGS. 2 to 4 show Jatropha curcas cultures with multiple shoots from asingle explant.

FIG. 5 shows Jatropha curcas shoots with roots.

FIG. 6 shows Jatropha curcas plantlets in greenhouse during thehardening step.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Micropropagation” refers to the in vitro regeneration of plants fromorgans, tissues, cells or protoplasts and the true-to-type propagationof a selected genotype using in vitro culture technique.

“Callus” refers to an unorganized or undifferentiated mass ofproliferative cells produced either in culture or in nature.

“True-to type propagation” means that all characteristics present inmother plant will also be present in next generation, i.e., theplantlets will be the true type of the mother plant.

“Fungicide” means any chemical substance that destroys and inhibits thegrowth of fungi. “Insecticide” means any substance, synthetic ororganic, which inhibits, kills, or destroys insects.

“MS” refers to Murashige and Skoog's medium.

“IBA” refers to Indole-3-butyric acid.

“FYM” refers to farm yard manure which can be like compost.

“M-45” refers to Dithane M-45.

“BAP” refers to 6-benzyl amino purine.

General

The present invention provides a commercially viable process for invitro mass culture of Jatropha curcas to generate true-to-type clones ofelite variety having the steps of using meristematic explants andculturing them in a media with a reduced concentration of phytohormones.In contrast to existing methods, the process allows the cultivation oftrue-to-type clones with a high success rate. Using this process, onecan obtain multiple shoot ratios up to 1:3, rooting success rates up to90%, hardening success rates up 70%, and field success rates up to 100%.

In some embodiments, the process has steps including, but not limitedto, selecting the healthy mother plants, treating the mother plant,isolating the explants from an elite variety of mother plants, cleaningof the explants, sterilizing the explants by primary and secondarysterilization, inoculating the explants on culture initiation medium,transferring the cultures to proliferation and elongation medium,transferring the elongated shoots to rooting medium, subjecting in vitrogrown plantlets to primary and secondary hardening, and transferring thehardened plantlets to fields.

Preparation of the Mother Plant

In certain embodiments, the mother plant from which the explants areharvested is subject to screening to identify healthy specimens and/ortreatment to either maintain a disease-free state or to treat existingdisease.

Health can be determined by assessing the plants for their size, weight,general growth, appearance, and absence of infection or contamination.J. curcas plants are commonly infected with “frogeye” (Cercospera spp.),insects of the order of Heteroptera and the golden flea beetle(Podagrica species).

Decontamination can be performed by spraying the plants with agents suchas fungicides, insecticides, pesticides or the like. Preferredfungicides for the pretreatment of the mother plant include Bavistin™,Captan™, Dithane™, Thiram™, Thiovit™, or combinations thereof at aconcentration of about 0.05% to 0.2%. Preferred insecticides for thepretreatment of the mother plant include, but are not limited to,Rogor™, Nuvacron, Fastac™, Ultracid™ 40 WP, Thiodane™ at a concentrationof about 0.005% to 0.02%.

Explants

The present invention provides a method for efficient in vitro massculture of Jatropha curcas using explants from meristematic tissue.Since meristematic cells are undifferentiated, the use of such tissue asan explant allows regeneration of true-to-type clones of the motherplants.

In preferred embodiments, shoot tip or nodal buds are used as explants.In the most preferred embodiments, the contemplated explant is shoot tipwith bud tissue. Apical meristem bud tissue is particularly preferred,as it is an active part of the plant and relatively contamination free.

Preferably, the explant used in the present invention is selected fromhealthy, fresh, disease-free plants. The explants may be isolated frommother plants growing in various locations, both wild and cultivated.

Preparation of the Explants for Culture Cleaning of Explants

In some embodiments, the explants are cleaned prior to inoculation inthe media. Cleaning is performed using methods known to those of skillin the art, for example, by shaking explants in a mild detergent, suchas Tween-20.

Sterilization of Explants

In other embodiments, the explants are sterilized prior to inoculationin the media. Sterilization can be performed using any method known tothose of skill in the art, for example, by treatment with fungicide, asurface sterilizing agent, or combinations thereof. The explant may besubjected to a single round of sterilization or multiple rounds ofsterilization.

For example, the explant may go through a primary sterilization stepwith the fungicide Bavistin and then go through a secondarysterilization with a surface sterilizing agent like sodium hypochloriteor mercuric chloride.

Culture of Explants

The present invention provides a method for efficient in vitro massculture of Jatropha curcas using meristematic explants and culture inmedia with a reduced concentration of phytohormones.

Other aspects of the micropropagation process can be performed usingmethods known to those of skill in the art in plant tissue culture.Micropropagation typically involves the following steps:

1) culturing explants in initiation media to generate multiple shoots

2) transferring shoots to proliferation and elongation media

3) transferring the elongated shoots to rooting media

4) hardening the plantlets, and

5) transferring the hardened plantlets to fields.

The basal media used to culture Jatropha can be any of those alreadyknown in the field of the art for plant tissue culture, such asMurashige & Skoog, Gamborg's, Vacin & Went, White's, Schenk &Hildebrandt or the like.

Basal media can also be supplemented with various carbon sources. Thecarbon source may be sucrose or glucose, typically, at a concentrationof about 2-5%. The carbon source may also be sugar alcohol likemyo-inositol, typically, at a concentration of about 50-500 mg perliter.

In some embodiments, the basal media will include gelling agents such asagar, alginic acid, carrageenan, gellangum. Typical concentrations are0.5-1%.

In one embodiment, the initiation medium is Murashige & Skoog mediumwith full strength of the basal nutrients with a reduced concentrationof phytohormones, such as 6-benzyl amino purine (BAP) in the range of0.44 μM-2.22 μM.

In some embodiments, the proliferation and shoot elongation medium androoting medium have the same level of nutrients and phytohormones as theinitiation medium. In other embodiments, the compositions are similarbut not precisely the same.

Phytohormones in Media

The present invention provides for a method where meristematic explantsare grown in media containing a reduced concentration of phytohormones.

The phytohormones used the media can be any phytohormone that willaffect growth in the desired manner during different stages of tissueculture. Examples of suitable phytohormones include natural or syntheticauxin, cytokinin, gibberellin, or cytokinin-active urea derivatives.

The cytokinins used can include, but are not limited to 6-aminopurine(adenine), 6-aminopurine hydrochloride, 6-aminopurine hemisulfate,6-benzyl aminopurine (BAP), kinetin, zeatin, N₆-substituted derivatives,or derivatives of these compounds. Preferred cytokinin-active ureaderivatives include, but are not limited to, thiadiziron, diphenylurea,N-phenyl-N′-(4-pyridyl) urea or their derivatives.

The auxins used can include, but are not limited to, naphthalene aceticacid, naphthaleneacetamide, naphthoxyacetic acid, indole acetic acid,indole butyric acid (IBA), 4-chlorophenoxyacetic acid,2,4-dichlorophenoxyacetic Acid (2,4-D), 2,4,5-trichlorophenoxyaceticacid, or the like and their derivatives.

The phytohormone may be used singly or in combination with two or moreother phytohormones.

The concentration of the phytohormone present in the media will bereduced as compared to that typically used to culture explants. Theexact concentration used will depend on the stage of the method of theinvention. The present invention provides that concentration is between0.01 mg per liter to 10 mg per liter, such as 0.1, 0.5, 1, or 5 mg/L. Inpreferred embodiments, there is a low level of cytokinin and auxin inthe media, for example, between 0.1 mg and 0.3 mg/L.

In one embodiment of this invention, a low level of cytokinin is used inthe initiation and proliferation/elongation media while a low level ofauxin is used in the rooting media.

In certain embodiments, the proliferation/elongation media also containsadenine sulphate, glutamine and activated charcoal.

Culture Conditions

In certain embodiments, the culture conditions (i.e., light cycle, lightintensity, media, temperature, relative humidity) are the samethroughout the initiation, proliferation and elongation, and rootingstages. Subculturing is performed as necessary; preferably, every 3 to 4weeks.

Once well-formed roots are obtained, plantlets can be hardened on soil,sand, moss, charcoal or other media either alone or in combination indefined ratio. The plantlets can then be transferred to the fields bydirect sowing or transplanting of the cuttings.

All references cited herein are hereby incorporated by reference.

The invention will be better understood by reference to the followingExample.

EXAMPLE

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Selection of Explant

The shoots of a healthy elite mother plant of Jatropha curcas from thefield were collected. Shoots were thoroughly washed under running waterto remove dust adhering to the shoot.

Cleaning of the Explant

All pieces were separately cleaned with a 0.5% Tween-20 solution withintermittent shaking for 5 minutes and then washed thoroughly withdemineralized water.

Sterilization of the Explant

Cleaned explants were subjected to primary sterilization by treating theexplants with a solution containing 0.1% Bavistin™ for 5 minutes andthen rinsing with sterile water. The explants were then subjected tosecondary sterilization in a laminar flow bench by treating with 0.5%sodium hypochlorite for 5 minutes and rinsing with autoclaved distilledwater repeatedly.

Preparation of the Explant for Inoculation

The explant was trimmed without damaging the apical and axillarymeristem to isolate the meristematic tissue.

To avoid the contamination and the resultant loss of valuable cultures,each explant was washed and treated separately.

Inoculation and Proliferation/Elongation (FIGS. 1, 2, 3, 4)

The sterilized explants were inoculated in Murashige & Skoog BasalMedium with 0.44 μM 6-benzyl amino purine. The explants were culturedunder the following conditions: an initial photoperiod of 16 hours under2000 lux light intensity followed by 8 hours dark period at 25° C.temperature and 60% RH. After multiple shoots were generated, they wereisolated and transferred into proliferation and elongation media withthe same composition as the initiation medium. The multiple shoot ratioobtained was around 1:3. The elongated shoots were subcultured at aregular interval of about 4 weeks.

Transferring to Rooting Medium (FIG. 5)

The healthy elongated shoots were transferred to rooting medium composedof half strength Murashige & Skoog Basal Medium with reducedconcentration of auxin, 4.9 μM IBA, which allowed the shoots to grow togive well-formed roots.

Hardening Protocol for Jatropha Plantlets in Greenhouse PrimaryHardening (FIG. 6)

The steps involved are described below:

-   -   1. The plantlets raised in vitro were washed in tap water and        then dipped in 1% Bavistin for 10 minutes.    -   2. They were then planted in portrays filled with FYM and soil        (1:1) treated with 1% M-45 solution.    -   3. These portrays were then kept under polytunnels for 20 days,        where a temperature of 23 to 28° C. and a relative humidity of        70% to 80% was maintained.    -   4. After 20 days, the plants were removed from polytunnels and        kept at a temperature of 25 to 30° C. and a relative humidity of        60% for 20 days

Secondary Hardening

The steps involved are described below:

-   -   1. The plants were shifted in polybags with a potting mixture        consisting of FYM and soil in a 1:1 ratio.    -   2. The plants were irrigated every three days.    -   3. After two months, the plants were ready to be dispatched to        the field.

Thus, while we have described fundamental novel features of theinvention, it will be understood that various omissions andsubstitutions and changes in the form and details may be possiblewithout departing from the spirit of the invention. For example, it isexpressly intended that all combinations of those elements and/ormethods steps, which perform substantially the same function insubstantially the same way to achieve the same results, be within thescope of the invention

1. A method for producing a true-to-type clone of a Jatropha curcasmother plant comprising culturing a meristematic explant of Jatrophacurcas in media with phytohormones at a concentration from about 0.01mg/L to about 10 mg/L and producing a true-to-type clone of a Jatrophacurcas mother plant from said meristematic explant.
 2. The method ofclaim 1, wherein said meristematic explant is from a shoot tip or anodal bud.
 3. The method of claim 2, wherein said meristematic explantis from a shoot tip.
 4. The method of claim 3, wherein said shoot tipcomprises bud tissue.
 5. The method of claim 4, wherein said bud tissueis apical bud tissue.
 6. The method of claim 1, wherein saidphytohormones are at concentration selected from the group consisting of0.01 mg/L, 0.1 mg/L, 0.5 mg/L, 1 mg/L, 5 mg/L, and 10 mg/L.
 7. Themethod of claim 1, wherein said phytohormones are at a concentrationfrom about 0.1 mg/L to about 0.3 mg/L.
 8. The method of claim 1, whereinsaid phytohormones are selected from the group consisting of cytokinins,cytokinin-active urea derivatives, auxins, and gibberellins.
 9. Themethod of claim 8, wherein said phytohormones are cytokinins.
 10. Themethod of claim 9, wherein said cytokinins are selected from the groupconsisting of 6-aminopurine (adenine), 6-aminopurine hydrochloride,6-aminopurine hemisulfate, 6-benzyl aminopurine (BAP), kinetin, zeatin,and N6-substituted derivatives.
 11. The method of claim 10, wherein saidcytokinin is 6-benzyl aminopurine.
 12. The method of claim 11, whereinsaid 6-benzyl aminopurine is at a concentration from about 0.44 μM toabout 2.22 μM.
 13. The method of claim 12, wherein said 6-benzylaminopurine is at a concentration of about 0.44 μM.
 14. The method ofclaim 8, wherein said phytohormones are cytokinin-active ureaderivatives.
 15. The method of claim 14, wherein said cytokinin-activeurea derivatives are selected from the group consisting of thiadiziron,diphenylurea, and N-phenyl-N′-(4-pyridyl) urea.
 16. The method of claim8, wherein said phytohormones are auxins.
 17. The method of claim 16,wherein said auxins are selected from the group consisting ofnaphthalene acetic acid, naphthaleneacetamide, naphthoxyacetic acid,indole acetic acid, indole butyric acid (IBA), 4-chlorophenoxyaceticacid, 2,4-dichlorophenoxyacetic acid (2,4-D), and2,4,5-trichlorophenoxyacetic acid.
 18. The method of claim 17, whereinsaid auxin is indole butyric acid.
 19. The method of claim 18, whereinsaid indole butyric acid is at a concentration of about 4.9 μM.